High-value oxy-pharmaceuticals from P450 BM3 'gatekeeper' mutations

Abstract

P450 BM3 is a natural fusion protein containing a heme catalytic domain fused to a CPR-like domain that binds two flavin cofactors (FAD and FMN). This allows this enzyme to have one of the highest catalytic rates of any P450. Such characteristics make P450 BM3 an attractive enzyme for mutagenesis to produce non-natural products. Unfortunately, no full-length structure revealing the natural domain interactions has been elucidated using X-ray crystallography or other means. HDX-MS is a relatively new technique to explore the surface of a protein and any solvent accessible channels, such as active sites and allosteric binding sites. HDX-MS and other MS techniques were utilized to probe the surface of WT P450 BM3 to gain new insights into the structure of the enzyme including potential dimeric interfaces. The observation of many shielded areas has given insights into conformational changes during ligand binding and on the dimeric interface of the protein, aiding in piecing together the full-length structure of this enzyme. WT BM3 binds specifically to fatty acids and typically produces hydroxylated products. Previously, two mutations were discovered that greatly affect the substrate and product profile of the double mutant (DM, A82F/F87V) enzyme. The substrate/ligand-binding profile of the DM enzyme was investigated using an FDA-approved library. From the library, 59% of the drugs caused significant shifts in the UV-Vis spectrum. These compounds have a variety of targets, masses and structures. Around 80 compounds were chosen due to their structural and binding properties for further analysis, including binding affinity determination, EPR, HPLC, LC-MS, LC-MS/MS, NMR and X-ray crystallography. From these analyzes, the binding of 18 inhibitors was identified, including a novel binding mode involving a substituted pyrimidine ring. Many substrates were also identified, which were found to be metabolized to known human metabolites during turnover.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)